The long term goal of this project is to understand, on a fundamental level, how the Tetrahymena ribozyme achieves its enormous rate enhancement and how this intron carries out the complex series of step required for the accurate and efficient ligation of exons. It is hoped that such in-depth understanding will further more general understanding of both biological catalysis and RNA. In addition, ribozymes are under investigation and potential therapeutics for the targeted destruction of specific RNAs in vivo, and it is possible that fundamental insights provided by this work will aid in the design of such therapeutic RNAs. Specific aims for the next five years are as follows: 1. The kinetic and thermodynamic framework for Tetrahymena ribozyme reactions will be extended to probe specific mechanistic questions of RNA catalysis and to address how this intron functions to carry out the multi-step self- splicing reaction. Such detailed analysis of individual reaction steps is crucial for dissecting catalytic strategies and for understanding function in complex molecular process such as self-splicing. The group I self-splicing reaction may provide a model for the involvement of RNA in more complex processes such as pre-mRNA splicing and translation. 2. Divalent metal ions are crucial to RNA folding and function, but the role of individual metal ions is typically obscured by the sea of metal ions that coat the charged phosphodiester backbone of RNA. The metal ions involved in the Tetrahymena ribozyme reaction will be studied in detail by dissecting active site interactions, determining catalytic roles, and characterizing the properties of these functional metal ion binding sites. 3. The use of noncovalent interactions to facilitate chemical transformations might be considered the most general hallmark of biological catalysts. The use of noncavalent interactions in the Tetrahymena ribozyme reactions will be probed on three levels: How does RNA use noncovalent interactions to fold and assemble an active site? What are the properties of the assembled active site? And how does the active site use binding energy for catalysis? In addition to revealing basic features of the energetic behavior of a large, structured RNA, it is hoped that these studies will provide insights into the fundamental interconnection of binding interactions and rate acceleration in biological catalysis.